The most general method for detecting the amplification product obtained by nucleic acid amplification such as polymerase chain reactions (PCR) is carried out by subjecting the solution after amplification to agarose gel electrophoresis and binding a fluorescent intercalator such as ethidium bromide thereto, and then observing specific fluorescence. When there is no possibility of contamination by other DNA and only the occurrence of the amplification product is of interest, fluorescence can be observed by adding a fluorescent intercalator to the solution after amplification while omitting electrophoresis. A fluorescent intercalator, however, binds to a single-stranded DNA such as a primer and emits fluorescence. Accordingly, a significant level of background noise can be contained in the detected fluorescent signal.
Recently, the present inventors have succeeded in developing a novel method for nucleic acid amplification, which does not require the complicated temperature control that is supposedly inevitable in PCR, i.e., the loop-mediated isothermal amplification (LAMP) method (Notomi, T. et al., Nucleic Acids Res. 28 (12), e63 (2000), WO 00/28082). In the LAMP method, the 3′ terminal region of template polynucleotide is self-annealed, synthesis of complementary strands is started therefrom, and a primer that is annealed to the loop formed in the aforementioned synthesis is used in combination therewith. This enables the amplification under isothermal conditions, and has remarkably enhanced the simplicity of nucleic acid amplification.
In real-time monitoring of the product of nucleic acid amplification using a fluorescent intercalator, fluorescence intensity significantly varies in PCR since the product of nucleic acid amplification is repeatedly dissociated and reassociated due to thermal denaturation as a thermal cycle proceeds. In the LAMP method, however, fluorescence intensity does not vary since the reaction proceeds under isothermal conditions. Thus, the LAMP method is more suitable for real-time monitoring of the product of nucleic acid amplification. The LAMP method, however, requires approximately 10 times as many primers as the quantity required in PCR. When the product of nucleic acid amplification obtained by the LAMP method is intended to be detected using a fluorescent intercalator, the level of background noise caused by single-stranded primers, which are also present therein, is high. Thus, it is difficult to detect only the amplified double-stranded nucleic acids with high sensitivity.
An object of the present invention is to provide a process for detecting nucleic acids that can detect double-stranded nucleic acids using an intercalator with higher sensitivity by reducing signals derived from an intercalator bound to single-stranded nucleic acids.
The present inventors have conducted concentrated studies in order to attain the above object. As a result, they have succeeded in reducing signals derived from an intercalator bound to a single-stranded nucleic acid with the addition of a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid or a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator to a mixture comprising double-stranded and single-stranded nucleic acids both having intercalators bound thereto. This has led to the completion of the present invention.
More specifically, the present invention relates to a method for reducing signals derived from an intercalator bound to a single-stranded nucleic acid, wherein a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid is added to a mixture comprising double-stranded and single-stranded nucleic acids both having intercalators (e.g., ethidium bromide, acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide, or YO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide) bound thereto, thereby reducing signals derived from an intercalator bound to a single-stranded nucleic acid. Examples of a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid include an oxidant, such as sodium hypochlorite, hydrogen peroxide, or potassium permanganate, and a reducer, such as sodium borohydride or sodium cyanoborohydride.
Further, the present invention relates to a method for reducing signals derived from an intercalator bound to a single-stranded nucleic acid, wherein a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is added to a mixture comprising double-stranded and single-stranded nucleic acids both having intercalators (e.g., ethidium bromide, acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide), or YO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide) bound thereto, thereby reducing signals derived from an intercalator bound to a single-stranded nucleic acid. An example of a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is a second intercalator (e.g. methylene blue, actinomycin D, SYBR® Green 2 (CAS Registry No. 172827-25-7), or OliGreen® (CAS Registry No. 268220-33-3)) different from the above intercalator.
Furthermore, the present invention relates to a method for detecting a product of nucleic acid amplification comprising the following-steps:
(a) amplifying a nucleic acid through nucleic acid amplification;
(b) adding an intercalator to a reaction solution after the nucleic acid amplification;
(c) reducing signals derived from an intercalator bound to a single-stranded nucleic acid by any of the aforementioned methods; and
(d) assaying the fluorescence intensity of a reaction solution.
Further, the present invention relates to a method for detecting a product of nucleic acid amplification comprising the following steps:
(a) amplifying a nucleic acid through nucleic acid amplification in the presence of an intercalator;
(b) reducing signals derived from an intercalator bound to a single-stranded nucleic acid by any of the aforementioned methods; and
(c) assaying the fluorescence intensity of a reaction solution.
The present invention further relates to a method for detecting a product of nucleic acid amplification comprising the following steps:
(a) amplifying a nucleic acid through nucleic acid amplification in the presence of an intercalator and a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator; and
(b) assaying the fluorescence intensity of a reaction solution.
The nucleic acid amplification can be carried out by the following steps:
(a) selecting a first arbitrary sequence F1c, a second arbitrary sequence F2c, and a third arbitrary sequence F3c in that order from the 3′ terminus in a target region toward the 3′ terminus on the polynucleotide chain and a fourth arbitrary sequence R1, a fifth arbitrary sequence R2, and a sixth arbitrary sequence R3 in that order from the 5′ terminus in the target region toward the 5′ terminus of the nucleotide chain;
(b) preparing a primer containing sequence F2 which is complementary to F2c and, on the 5′ side of F2, the same sequence as F1c; a primer containing sequence F3 which is complementary to F3c; a primer containing the same sequence as R2 and, on the 5′ side of the sequence, sequence R1c which is complementary to R1; and a primer containing the same sequence as R3; and
(c) synthesizing DNA in the presence of a strand displacement-type polymerase and the primers using the nucleotide chain as a template.
The nucleic acid amplification can be carried out by the following steps:
(a) selecting a first arbitrary sequence F1c and a second arbitrary sequence F2c in that order from the 3′ terminus in a target region toward the 3′ terminus on the polynucleotide chain and a third arbitrary sequence R1 and a fourth arbitrary sequence R2 in that order from the 5′ terminus in the target region toward the 5′ terminus of the nucleotide chain;
(b) preparing a primer containing sequence F2 which is complementary to F2c and, on the 5′ side of F2, the same sequence as F1c; and a primer containing the same sequence as R2 and, on the 5′ side of the sequence, sequence R1c which is complementary to R1; and
(c) synthesizing DNA in the presence of a strand displacement-type polymerase, the primers, and a melting temperature regulator (such as betaine or trimethylamine N-oxide) using the nucleotide chain as a template for amplification.
The present invention further relates to a kit for detecting double-stranded nucleic acids comprising, as elements, an intercalator (e.g, ethidiun, bromide, acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide), or YO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide) and a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid and/or a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator. Examples of the compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid include an oxidant such as sodium hypochlorite, hydrogen peroxide, or potassium permanganate, and a reducer, such as sodium borohydride or sodium cyanoborohydride. An example of the compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is a second intercalator (e.g. methylene blue, actinomycin D, SYBR® Green 2 (CAS Registry No. 172827-25-7), or OliGreen® (CAS Registry No. 268220-33-3)) different from the aforementioned intercalator.